Muneera J. Alshammari

Study of autosomal recessive osteogenesis imperfecta in Arabia reveals a novel locus defined by TMEM38B mutation

Background Osteogenesis imperfecta (OI) is an hereditary bone disease in which increased bone fragility leads to frequent fractures and other complications, usually in an autosomal dominant fashion. An expanding list of genes that encode proteins related to collagen metabolism are now recognised as important causes of autosomal recessive (AR) OI. Our aim was to study the contribution of known genes to AR OI in order to identify novel loci in mutation-negative cases. Methods We enrolled multiplex consanguineous families and simplex cases (also consanguineous) in which mutations in COL1A1 and COL1A2 had been excluded. We used autozygome guided mutation analysis of AR OI (AR OI) genes followed by exome sequencing when such analysis failed to identify the causative mutation. Results Two simplex and 11 multiplex families were enrolled, encompassing 27 cases. In three multiplex families, autozygosity and linkage analysis revealed a novel recessive OI locus on chromosome 9q31.1-31.3, and a novel truncating deletion of exon 4 of TMEM38B was identified within that interval. In addition, gonadal or gonadal/somatic mosaic mutations in COL1A1 or COL1A2 and homozygous mutations in recently described AR OI genes were identified in all remaining families. Conclusions TMEM38B is a novel candidate gene for AR OI. Future studies are needed to explore fully the contribution of this gene to AR OI in other populations.

Genomic analysis of mitochondrial diseases in a consanguineous population reveals novel candidate disease genes

Objective To investigate the utility of autozygome analysis and exome sequencing in a cohort of patients with suspected or confirmed mitochondrial encephalomyopathy. Methods Autozygome was used to highlight candidate genes for direct sequencing in 10 probands, all born to consanguineous parents. Autozygome was also used to filter the variants from exome sequencing of four probands. Results In addition to revealing mutations in known mitochondrial genes, the analysis revealed the identification of two novel candidate disease genes: MFF and FARS2, encoding the mitochondrial fission factor and phenylalanyl-tRNA synthetase, respectively. Interpretation These findings expand the repertoire of genes that are mutated in patients with mitochondrial disorders and highlight the value of integrating genomic approaches in the evaluation of these patients.

POC1A Truncation Mutation Causes a Ciliopathy in Humans Characterized by Primordial Dwarfism

Primordial dwarfism (PD) is a phenotype characterized by profound growth retardation that is prenatal in onset. Significant strides have been made in the last few years toward improved understanding of the molecular underpinning of the limited growth that characterizes the embryonic and postnatal development of PD individuals. These include impaired mitotic mechanics, abnormal IGF2 expression, perturbed DNA-damage response, defective spliceosomal machinery, and abnormal replication licensing. In three families affected by a distinct form of PD, we identified a founder truncating mutation in POC1A. This gene is one of two vertebrate paralogs of POC1, which encodes one of the most abundant proteins in the Chlamydomonas centriole proteome. Cells derived from the index individual have abnormal mitotic mechanics with multipolar spindles, in addition to clearly impaired ciliogenesis. siRNA knockdown of POC1A in fibroblast cells recapitulates this ciliogenesis defect. Our findings highlight a human ciliopathy syndrome caused by deficiency of a major centriolar protein.

Genomic analysis of Meckel–Gruber syndrome in Arabs reveals marked genetic heterogeneity and novel candidate genes

Meckel–Gruber syndrome (MKS, OMIM #249000) is a multiple congenital malformation syndrome that represents the severe end of the ciliopathy phenotypic spectrum. Despite the relatively common occurrence of this syndrome among Arabs, little is known about its genetic architecture in this population. This is a series of 18 Arab families with MKS, who were evaluated clinically and studied using autozygome-guided mutation analysis and exome sequencing. We show that autozygome-guided candidate gene analysis identified the underlying mutation in the majority (n=12, 71%). Exome sequencing revealed a likely pathogenic mutation in three novel candidate MKS disease genes. These include C5orf42, Ellis–van-Creveld disease gene EVC2 and SEC8 (also known as EXOC4), which encodes an exocyst protein with an established role in ciliogenesis. This is the largest and most comprehensive genomic study on MKS in Arabs and the results, in addition to revealing genetic and allelic heterogeneity, suggest that previously reported disease genes and the novel candidates uncovered by this study account for the overwhelming majority of MKS patients in our population.

Molecular characterization of Joubert syndrome in Saudi Arabia.

Joubert syndrome (JS) is a ciliopathy that is defined primarily by typical cerebellar structural and ocular motility defects. The genetic heterogeneity of this condition is significant with 16 genes identified to date. We have used a combination of autozygome‐guided candidate gene mutation analysis and exome sequencing to identify the causative mutation in a series of 12 families. The autozygome approach identified mutations in RPGRIP1L, AHI1, TMEM237, and CEP290, while exome sequencing revealed families with truncating mutations in TCTN1 and C5ORF42. Our study, the largest comprehensive molecular series on JS, provides independent confirmation of the recently reported TCTN1, TMEM237, and C5ORF42 as bona fide JS disease genes, and expands the allelic heterogeneity of this disease. Hum Mutat 33:1423–1428, 2012.

Mutations in MEOX1, encoding mesenchyme homeobox 1, cause Klippel-Feil anomaly.

Klippel-Feil syndrome (KFS) is a segmentation malformation of the cervical spine; clinically, it manifests as a short neck with reduced mobility and a low posterior hairline. Several genes have been proposed as candidates for KFS when it is present with other associated anomalies, but the genetics of isolated KFS have been difficult to study because of the syndromes mostly sporadic occurrence. We describe a multiplex consanguineous family in which isolated KFS maps to a single 17q21.31 locus that harbors a homozygous frameshift deletion in MEOX1; this deletion results in complete instability of the transcript. Direct sequencing of this gene in two siblings from another consanguineous family affected by isolated KFS uncovered another homozygous truncating (nonsense) MEOX1 mutation that also leads to complete degradation of the transcript. This gene encodes a transcription factor with a well-established and nonredundant role in somite development, and homozygous null alleles of Meox1 in mice have a cervical skeletal defect that is remarkably similar to the one we observe in human individuals with MEOX1 mutations. Our data strongly suggest that KFS is the human phenotypic equivalent of the sclerotome polarity defect that results from Meox1 deficiency in mice.

A novel syndrome of hypohidrosis and intellectual disability is linked to COG6 deficiency.

Background Numerous syndromic forms of intellectual disability have been described including those with abnormal sweating pattern. Purpose To describe the clinical and molecular analysis of a large multiplex consanguineous Saudi family with an unusual constellation of severe intellectual disability, hypohidrosis, abnormal teeth, and acquired microcephaly. Methods Clinical evaluation, autozygosity mapping, exome sequencing, and expression analysis. Results Autozygosity mapping revealed a single critical locus corresponding to chr13:39 338 062–40 857 430. Exome sequencing uncovered a deep intronic (NM_020751.2:c.1167–24A>G) variant in COG6 that largely replaces the consensus acceptor site, resulting in pronounced reduction of the normal transcript and consequent deficiency of COG6 protein. Patient cells also exhibited pronounced deficiency of STX6, consistent with the established stabilising effect of COG6 on STX6. Four additional patients representing two families of the same tribal origin as the original family were found to have the same mutation, confirming a founder effect. Remarkably, none of the patients displayed any detectable abnormality in the glycosylation pattern of transferrin, which contradicts a previously published report of a patient whose abnormal glycosylation pattern was presumed to be caused by a missense variant in COG6. Conclusions Our data implicate COG6 in the pathogenesis of a novel hypohidrotic disorder in humans that is distinct from congenital disorders of glycosylation.

The Syndrome of Microcornea, Myopic Chorioretinal Atrophy, and Telecanthus (MMCAT) Is Caused by Mutations in ADAMTS18

One of us recently described an apparently novel ocular syndrome characterized by microcornea, myopic chorioretinal atrophy, and telecanthus (MMCAT) in a number of Saudi families. Consistent with the presumed pseudodominant inheritance in one of the original families, we show that MMCAT maps to a single autozygous locus on chr16q23.1 in which exome sequencing revealed a homozygous missense change in ADAMTS18. Direct sequencing of this gene in four additional probands with the same phenotype revealed three additional homozygous changes in ADAMTS18 including two nonsense mutations. Reassuringly, the autozygomes of all probands overlap on the same chr16q23.1 locus, further supporting the positional mapping of MMCAT to ADAMTS18. ADAMTS18 encodes a member of a family of metalloproteinases that are known for their role in extracellular matrix remodeling, and previous work has shown a strong expression of Adamts18 in the developing eye. Our data suggest that ADAMTS18 plays an essential role in early eye development and that mutations therein cause a distinct eye phenotype that is mainly characterized by microcornea and myopia.

Expanding the clinical and genetic heterogeneity of hereditary disorders of connective tissue.

Ehlers–Danlos syndrome (EDS) describes a group of clinical entities in which the connective tissue, primarily that of the skin, joint and vessels, is abnormal, although the resulting clinical manifestations can vary widely between the different historical subtypes. Many cases of hereditary disorders of connective tissue that do not seem to fit these historical subtypes exist. The aim of this study is to describe a large series of patients with inherited connective tissue disorders evaluated by our clinical genetics service and for whom a likely causal variant was identified. In addition to clinical phenotyping, patients underwent various genetic tests including molecular karyotyping, candidate gene analysis, autozygome analysis, and whole-exome and whole-genome sequencing as appropriate. We describe a cohort of 69 individuals representing 40 families, all referred because of suspicion of an inherited connective tissue disorder by their primary physician. Molecular lesions included variants in the previously published disease genes B3GALT6, GORAB, ZNF469, B3GAT3, ALDH18A1, FKBP14, PYCR1, CHST14 and SPARC with interesting variations on the published clinical phenotypes. We also describe the first recessive EDS-like condition to be caused by a recessive COL1A1 variant. In addition, exome capture in a familial case identified a homozygous truncating variant in a novel and compelling candidate gene, AEBP1. Finally, we also describe a distinct novel clinical syndrome of cutis laxa and marked facial features and propose ATP6V1E1 and ATP6V0D2 (two subunits of vacuolar ATPase) as likely candidate genes based on whole-genome and whole-exome sequencing of the two families with this new clinical entity. Our study expands the clinical spectrum of hereditary disorders of connective tissue and adds three novel candidate genes including two that are associated with a highly distinct syndrome.

Mutations in C12orf57 Cause a Syndromic Form of Colobomatous Microphthalmia

Microphthalmia is an important developmental eye disorder. Although mutations in several genes have been linked to this condition, they only account for a minority of cases. We performed autozygome analysis and exome sequencing on a multiplex consanguineous family in which colobomatous microphthalmia is associated with profound global developmental delay, intractable seizures, and corpus callosum abnormalities, and we identified a homozygous truncating mutation in C12orf57 [c.1A>G; p.Met1?]. In a simplex case with a similar phenotype, we identified compound heterozygosity for the same mutation and another missense mutation [c.152T>A; p.Leu51Gln]. Little is known about C12orf57 but we show that it is expressed in several mouse tissues, including the eye and brain. Our data strongly implicate mutations in C12orf57 in the pathogenesis of a clinically distinct autosomal-recessive syndromic form of colobomatous microphthalmia.